Chromosomal multidrug resistance (MDR) in bacteria is a serious clinical problem. Our studies have shown that Escherichia coli becomes resistant to a variety of antibiotics, organic solvents and superoxides when the activities of any of three paralogous, but differently regulated, transcriptional activators, MarA, SoxS and Rob, are increased. These activators bind a sequence called the marbox which lies upstream of the promoters of a set of about 40 chromosomal genes called the marA/soxS/rob regulon. tolC and acrAB are regulon genes that have critical functions in multiple antibiotic resistance since their protein products constitute the most important MDR pump in E. coli. The major goals of this project are to understand the regulation of these activators, the mechanisms whereby they activate the regulon promoters, and the mechanisms whereby the MDR is generated. Upregulation of the transcriptional activators, MarA, SoxS and Rob can be effected by treating the cells with certain chemicals. Phenolic compounds derepress the marRAB operon;superoxides activate SoxR which in turn activates SoxS;and bile salts and other compounds activate the Rob protein directly. Thus, the upregulation of these activators can indicate the presence of such substances in the environment or in the cell. Previously, we found that E. coli tolC mutants, which do not have the TolC outer membrane channel, have elevated levels of transcription of marRAB and soxS and have elevated activity of Rob protein. Since TolC is a vital component of eight known efflux pumps in E. coli and plays important roles in ridding bacteria of multiple antibiotics, bile salts, organic solvents and other xenobiotics, we concluded (1) that in the absence of TolC, intracellular metabolic waste products accumulate and trigger the upregulation of the activators and (2) that TolC is normally involved in the efflux of cellular metabolites and not merely of xenobiotics. In collaboration with V. Schlegel, we have used Fourier transform infrared spectroscopy (FTIR) to compare the metabolome of tolC mutants with that of wild-type with the aim of identifying the activator-triggering metabolites that accumulate in tolC mutants. Several significant differences between the mutant and wild-type have now been found in the cellular constituents and in the post-growth extracellular medium. This supports our hypothesis that the loss of pumping function in the tolC mutants alters the composition of the cell and of the external environment. We have also identified three pumps that participate with TolC in pumping out the triggering metabolites. We used knock-out mutations, singly and in combination, to test the effects of the eight pumps known to require TolC as their external duct. Of the single knockouts, only the loss of AcrB had a measurable effect on activator upregulation. However, when both AcrB and EmrB were knocked out, the effect was substantially increased. Finally, the knockout of MdtE combined with knockouts of both AcrB and EmrB resulted in an effect on the activators comparable to that of the knockout of TolC. This suggests that the AcrAB-TolC pump makes the largest contribution to efflux of wastes and that EmrAB-TolC and MdtEF-TolC make lesser contributions by themselves. These results provide additional support for our hypothesis that E. coli pumps are used to excrete toxic metabolic wastes from the cell. We suggest that excretion of wastes may have been the original function of the MDR pumps.